CN101691330A - Separation and purification methods of highly purified antiviral active components in artichoke - Google Patents
Separation and purification methods of highly purified antiviral active components in artichoke Download PDFInfo
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- CN101691330A CN101691330A CN200910044426A CN200910044426A CN101691330A CN 101691330 A CN101691330 A CN 101691330A CN 200910044426 A CN200910044426 A CN 200910044426A CN 200910044426 A CN200910044426 A CN 200910044426A CN 101691330 A CN101691330 A CN 101691330A
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- China
- Prior art keywords
- caffeoylquinic acids
- caffeoylquinic
- caffeoylquinic acid
- acid
- water
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Abstract
本发明公开了一种高效分离制备洋蓟中高纯度抗病毒活性组分(单咖啡酰奎宁酸类化合物以及二咖啡酰奎宁酸类化合物)的方法，主要包括使用醇水溶液提取洋蓟鲜品或干品，浓缩后进行柱层析分离，制备富含抗病毒活性组分的粗提物，然后对粗提物再以高速逆流色谱结合高效制备液相色谱进行分离纯化，最后采用重结晶的方法进行精制，得到高纯度的1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸，5-O-咖啡酰奎宁酸，1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸。该方法适用于以各种途径获得的各种含以上咖啡酰奎宁酸类化合物中的一种或几种成分的天然产物或天然产物提取物为原料制备高纯度单体，并且步骤简单，操作简便，效率高，成本低，易于重复，分离制备量大。使用该方法制备的咖啡酰奎宁酸类化合物的纯度可达98％。The invention discloses a method for efficiently separating and preparing high-purity antiviral active components (monocaffeoylquinic acid compounds and dicaffeoylquinic acid compounds) in artichokes, which mainly includes extracting fresh artichokes with an aqueous alcohol solution Or dry product, concentrated and separated by column chromatography to prepare a crude extract rich in antiviral active components, then separate and purify the crude extract by high-speed countercurrent chromatography combined with high-efficiency preparative liquid chromatography, and finally use recrystallized The method is refined to obtain high-purity 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 1, 3-Di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid quinic acid and 4,5-di-O-caffeoylquinic acid. The method is suitable for preparing high-purity monomers from various natural products or natural product extracts containing one or several components of the above caffeoylquinic acid compounds obtained in various ways, and the steps are simple and easy to operate. The method is simple, high in efficiency, low in cost, easy to repeat, and large in amount of separation and preparation. The purity of the caffeoylquinic acid compounds prepared by the method can reach 98%.
Description
技术领域technical field
本发明涉及中药或天然产物中有效成分的分离纯化方法，主要涉及从中药或天然产物中提取、分离单体化合物的方法，具体涉及一种高纯度咖啡酰奎宁酸类化合物1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸，5-O-咖啡酰奎宁酸，1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸的分离制备方法。The present invention relates to a separation and purification method of effective components in traditional Chinese medicine or natural products, mainly relates to a method for extracting and separating monomeric compounds from traditional Chinese medicine or natural products, in particular to a high-purity caffeoylquinic acid compound 1-O-coffee Caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 1,3-di-O-caffeoylquinic acid, 1 , 5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid A method for the separation and preparation of acylquinic acid.
背景技术Background technique
洋蓟(Cynara scolymus L.，又名菜蓟、朝鲜蓟、洋百合、法国百合、荷花百合)系菊科多年生草本植物。原产地中海沿岸。我国在20世纪90年代从国外引种，目前湖南、浙江和云南等地均有大规模的种植。洋蓟的花蕾(肉质花托和总苞片)、茎叶自古药食两用，具有丰富的食用和生理活性价值，传统用于消化不良、肝胆疾患、利尿解毒、降脂、降压等。药理研究表明，洋蓟中含有多种抗病毒活性组分，如单咖啡酰奎宁酸类化合物(1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸和5-O-咖啡酰奎宁酸)以及二咖啡酰奎宁酸类化合物(1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸)等。Artichoke (Cynara scolymus L., also known as artichoke, artichoke, artichoke, French lily, lotus lily) is a perennial herb of Compositae. Native to the Mediterranean coast. my country introduced the species from abroad in the 1990s, and it is currently planted on a large scale in Hunan, Zhejiang, and Yunnan. Artichoke flower buds (succulent receptacles and involucral bracts), stems and leaves have been used for both medicine and food since ancient times. They have rich edible and physiological activity values. Pharmacological studies have shown that artichoke contains a variety of antiviral active components, such as monocaffeoylquinic acid compounds (1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O- Caffeoylquinic acid and 5-O-caffeoylquinic acid) and dicaffeoylquinic acid compounds (1,3-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid quinic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid) etc.
咖啡酰奎宁酸类化合物是由奎尼酸和咖啡酸缩合而成的一类多酚类化合物，广泛存在于植物界中，广为分布的单咖啡酰奎宁酸类化合物1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸和5-O-咖啡酰奎宁酸为同分异构体，此类化合物的区别在于一分子的咖啡酰基在奎宁酸上的连接位置不同，分子式均为C16H18O9；而二咖啡酰基奎宁酸类化合物为1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸也互为同分异构体，五者之间的的区别在于两个咖啡酰基在奎宁酸上的连接位置不同，分子式均为C25H24O12，分子量为516。结构式如下：Caffeoylquinic acid compounds are a class of polyphenolic compounds formed by the condensation of quinic acid and caffeic acid, which widely exist in the plant kingdom, and the widely distributed monocaffeoylquinic acid compound 1-O-coffee Acylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid and 5-O-caffeoylquinic acid are isomers, the difference of these compounds is that one molecule The connection position of caffeoyl group on quinic acid is different, and the molecular formula is C 16 H 18 O 9 ; while the dicaffeoyl quinic acid compound is 1,3-di-O-caffeoyl quinic acid, 1,5- Di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid The acids are also isomers of each other. The difference between the five lies in the connection position of the two caffeoyl groups on the quinic acid. The molecular formula is C 25 H 24 O 12 and the molecular weight is 516. The structural formula is as follows:
R1＝咖啡酰基，R2＝R3＝R4＝H 1-O-咖啡酰奎宁酸R 1 = caffeoyl, R 2 = R 3 = R 4 = H 1-O-caffeoylquinic acid
R2＝咖啡酰基，R1＝R3＝R4＝H 3-O-咖啡酰奎宁酸R 2 = caffeoyl, R 1 = R 3 = R 4 = H 3-O-caffeoylquinic acid
R3＝咖啡酰基，R1＝R2＝R4＝H 4-O-咖啡酰奎宁酸R 3 = caffeoyl, R 1 = R 2 = R 4 = H 4-O-caffeoylquinic acid
R4＝咖啡酰基，R1＝R2＝R3＝H 5-O-咖啡酰奎宁酸(绿原酸)R 4 = caffeoyl, R 1 = R 2 = R 3 = H 5-O-caffeoylquinic acid (chlorogenic acid)
R1＝R2＝咖啡酰基，R3＝R4＝H 1，3-二-O-咖啡酰奎宁酸(洋蓟酸)R 1 =R 2 =caffeoyl, R 3 =R 4 =H 1,3-di-O-caffeoylquinic acid (cynic acid)
R1＝R4＝咖啡酰基，R2＝R3＝H 1，5-二-O-咖啡酰奎宁酸R 1 =R 4 =caffeoyl, R 2 =R 3 =H 1,5-di-O-caffeoylquinic acid
R1＝R4＝咖啡酰基，R2＝R3＝H 1，5-二-O-咖啡酰奎宁酸R 1 =R 4 =caffeoyl, R 2 =R 3 =H 1,5-di-O-caffeoylquinic acid
R2＝R4＝咖啡酰基，R1＝R3＝H 3，5-二-O-咖啡酰奎宁酸R 2 =R 4 =caffeoyl, R 1 =R 3 =H 3,5-di-O-caffeoylquinic acid
R2＝R3＝咖啡酰基，R1＝R4＝H 3，4-二-O-咖啡酰奎宁酸R 2 =R 3 =caffeoyl, R 1 =R 4 =H 3,4-di-O-caffeoylquinic acid
R3＝R4＝咖啡酰基，R1＝R2＝H 4，5-二-O-咖啡酰奎宁酸R 3 =R 4 =caffeoyl, R 1 =R 2 =H 4,5-di-O-caffeoylquinic acid
近代药理学试验研究表明：咖啡酰奎宁酸类化合物具有多种重要生理活性，主要是针对免疫系统的作用，抑制乙肝病毒、艾滋病毒、单纯疱疹病毒及流感病毒，抑制白三烯合成和释放，抑制组胺释放，从而可用于抗病毒、抗炎和治疗过敏性疾病。此外还发现其具有强效的抗氧化作用、抑制脂氧酶抗动脉粥硬化作用、抗血小板聚集活性以及降血脂作用等活性。Modern pharmacological experiments have shown that caffeoylquinic acid compounds have a variety of important physiological activities, mainly targeting the immune system, inhibiting hepatitis B virus, HIV, herpes simplex virus and influenza virus, and inhibiting the synthesis and release of leukotrienes , inhibit the release of histamine, which can be used for antiviral, anti-inflammatory and treatment of allergic diseases. In addition, it has been found that it has potent anti-oxidation, lipoxygenase inhibition, anti-atherosclerosis, anti-platelet aggregation, and blood lipid-lowering effects.
咖啡酰奎宁酸类化合物为白色粉末状固体，易溶于热水、热甲醇、热乙醇，不溶于乙醚、苯等极性较小的有机溶剂，受热发生氧化分解或转化为其它二咖啡酰奎宁酸类化合物，此外，三个化合物在溶液中不稳定，能够相互转化，这给它们的分离纯化造成很大的困难。因此，建立一种高效、快速的从中药或天然药物中分离纯化高纯度的多个咖啡酰奎宁酸类化合物，对开发具有特殊疗效的现代中药制剂和充分利用我国的药材资源等具有重要意义。Caffeoylquinic acid compounds are white powdery solids, easily soluble in hot water, hot methanol, hot ethanol, insoluble in ether, benzene and other less polar organic solvents, oxidative decomposition or conversion into other dicaffeoyl quinic acid compounds when heated Quinic acid compounds, in addition, the three compounds are unstable in solution and can be transformed into each other, which causes great difficulties in their separation and purification. Therefore, the establishment of an efficient and rapid separation and purification of multiple high-purity caffeoylquinic acid compounds from traditional Chinese medicine or natural medicine is of great significance for the development of modern Chinese medicine preparations with special curative effects and the full utilization of medicinal resources in my country. .
目前通用的咖啡酰奎宁酸类化合物分离提取方法是用甲醇、乙醇或水回流提取，减压浓缩分离提取液得到浸膏，将浸膏悬浮于水，依次用石油醚或正己烷、氯仿或乙酸乙酯、水饱和的正丁醇等不同极性的溶剂萃取。咖啡酰奎宁酸类化合物一般集中在正丁醇相，再利用正相硅胶柱层析，使用石油醚、乙酸乙酯、氯仿、甲醇等溶剂洗脱，以及过Sephadex LH-20柱色谱或制备液相色谱得到单一化合物。这些方法步骤复杂，费时费力，而且回收率低，成本高，不适合工业化。高速逆流色谱(high speed counter current chromatography，HSCCC)是20世纪80年代初期发展起来的一种无需使用任何固态支持介质的液-液分配色谱技术，其基本原理是根据被分离的物质在两相溶剂系统中的分配系数不同而得到最终分离；这种分离技术由于不使用固体支持介质，避免了因与固体填料发生不可逆吸附而造成的样品损失、失活变性等常规填料易产生的弊端，可使样品得以全部回收，且具有分离容量大、性能强、高效、快速的特点，尤其适用于制备性分离，逆流色谱已广泛应用于生物、医药、环保等领域化学物质的制备分离和纯化。逆流色谱技术的发展和应用结合高效制备液相色谱高效的分离纯化能力解决了目前咖啡酰奎宁酸类化合物分离纯化技术面临的难题。The current general separation and extraction method of caffeoylquinic acid compounds is to use methanol, ethanol or water to reflux extraction, concentrate and separate the extract under reduced pressure to obtain an extract, suspend the extract in water, and use petroleum ether or n-hexane, chloroform or Ethyl acetate, water-saturated n-butanol and other solvents of different polarities. Caffeoylquinic acid compounds are generally concentrated in the n-butanol phase, then use normal phase silica gel column chromatography, use petroleum ether, ethyl acetate, chloroform, methanol and other solvents to elute, and pass through Sephadex LH-20 column chromatography or prepare Liquid chromatography yielded a single compound. These methods are complicated in steps, time-consuming and labor-intensive, and have low recovery rate and high cost, so they are not suitable for industrialization. High speed counter current chromatography (HSCCC) is a liquid-liquid partition chromatography technology developed in the early 1980s without using any solid support medium. The distribution coefficient in the system is different to obtain the final separation; this separation technology avoids the disadvantages of conventional fillers such as sample loss and inactivation denaturation caused by irreversible adsorption with solid fillers because it does not use solid support media. All samples can be recovered, and it has the characteristics of large separation capacity, strong performance, high efficiency and fast speed, especially suitable for preparative separation. Countercurrent chromatography has been widely used in the preparation, separation and purification of chemical substances in the fields of biology, medicine and environmental protection. The development and application of countercurrent chromatography combined with the high-efficiency separation and purification capabilities of high-efficiency preparative liquid chromatography have solved the current difficulties in the separation and purification of caffeoylquinic acid compounds.
发明内容Contents of the invention
本发明要解决的技术问题是克服传统分离技术的缺陷，提供一种从粗提物中分离纯化1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸，5-O-咖啡酰奎宁酸，1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸的制备方法，该方法具有操作简便，分离量大，回收率高等优点。The technical problem to be solved in the present invention is to overcome the defects of traditional separation techniques and provide a method for separating and purifying 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-coffee Acylquinic acid, 5-O-caffeoylquinic acid, 1,3-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, 3,5-di-O - the preparation method of caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid, the method has the advantages of simple operation, large separation capacity and high recovery rate Advanced advantages.
本发明的技术方案主要包括：Technical scheme of the present invention mainly comprises:
a)粗提物的制备a) Preparation of crude extract
将洋蓟植物粉碎，用水-强极性的有机溶剂提取，提取液减压浓缩至无醇味，并经离心去除杂质后得到澄清的浓缩液，进行大孔吸附树脂柱层析，先以水洗至近无色，再以不同浓度的乙醇水溶液梯度洗脱，接收50～70％的乙醇洗脱物，将洗脱液减压浓缩至无醇味，加入乙醇至含醇量70～80％，除去沉淀，取上清液浓缩，得含咖啡酰奎宁酸类化合物的粗品。Crush the artichoke plant, extract it with water-strong polar organic solvent, concentrate the extract under reduced pressure until it has no alcohol smell, and centrifuge to remove impurities to obtain a clear concentrate, perform macroporous adsorption resin column chromatography, and wash with water first until almost colorless, then elute with gradient ethanol aqueous solution of different concentrations, receive 50-70% ethanol eluate, concentrate the eluate under reduced pressure until it has no alcohol smell, add ethanol until the alcohol content is 70-80%, remove precipitation, and the supernatant was concentrated to obtain a crude product containing caffeoylquinic acid compounds.
b)目标化合物的分离纯化b) Separation and purification of the target compound
将富含咖啡酰奎宁酸类化合物的粗提物真空干燥后，用高速逆流色谱结合高效制备液相色谱进行分离制备，根据紫外检测谱图或者TLC图谱收集目标组份。After the crude extract rich in caffeoylquinic acid compounds is vacuum-dried, it is separated and prepared by high-speed countercurrent chromatography combined with high-performance preparative liquid chromatography, and the target components are collected according to the ultraviolet detection spectrum or TLC spectrum.
目标组分经真空干燥后，用有机溶剂或水-有机溶剂的混合液进行重结晶即可得高纯度的1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸，5-O-咖啡酰奎宁酸，1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸单体化合物。After the target components are vacuum-dried, recrystallize with an organic solvent or a water-organic solvent mixture to obtain high-purity 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4- O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 1,3-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, 3,5- Di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid monomer compounds.
制备方法中，步骤a)中所用原料优选菊科洋蓟；提取溶剂为醇水混合溶剂，其中醇可以是甲醇、乙醇、丙醇、丁醇、乙二醇等或它们的混合物，优选甲醇或乙醇，醇水混合溶剂可以是醇和水以任何比例混合的溶剂，优选乙醇和水混合溶剂，其间优选70～80％的乙醇水溶液。提取溶剂的重量为药材重量的1～20倍，优选6～10倍；提取时间为0.5-5小时，优选2小时；提取次数为1-5次，优选3次；柱层析所用介质为大孔吸附树脂、聚酰胺、反相硅胶或葡聚糖凝胶类，优选D101，AB-8，HPD100，HPD300，D1或D2型大孔吸附树脂，洗脱溶剂优选30～40％乙醇水溶液；步骤b)中，优选脂肪酯-脂肪醇-水或者卤代烃-脂肪醇-水组成的溶剂体系进行高速逆流色谱，脂肪酯可以是乙酸乙酯、乙酸丁酯、乙酸丙酯、甲酸乙酯，最优选为乙酸乙酯，卤代烃可以是氯仿、二氯甲烷或四氯甲烷，最优选为氯仿，脂肪醇可以是甲醇、乙醇、异丙醇或正丁醇，最优选为甲醇，其中主机转速正转为600～1200rpm，优选800rpm，流动相泵入柱内的流速为0.5～5mL/min，优选1.5mL/min，实验条件温度为10～30℃，优选为25℃，上相为固定相，下相为流动相；优选脂肪醇-水-酸或者脂肪腈-水-酸组成的溶剂体系进行高效液相色谱分离制备，脂肪醇可以是甲醇、乙醇等，最优选为甲醇，脂肪腈最优选为乙腈，酸可以是甲酸、乙酸、磷酸盐缓冲溶液等，最优选为乙酸，其中流速为5～20mL/min，优选10mL/min，实验条件温度为10～30℃，优选为25℃，采用梯度洗脱的方式；由于咖啡酰奎宁酸类化合物的不稳定性，分离和收集到的目标成分，必须立即真空干燥后重结晶，重结晶溶剂为有机溶剂或水-有机溶剂的混合液，优选80％甲醇水溶液或80％乙醇水溶液。In the preparation method, the raw material used in step a) is preferably Compositae artichoke; the extraction solvent is a mixed solvent of alcohol and water, wherein the alcohol can be methanol, ethanol, propanol, butanol, ethylene glycol, etc. or their mixtures, preferably methanol or Ethanol, the mixed solvent of alcohol and water can be the solvent that alcohol and water are mixed in any proportion, preferably ethanol and water mixed solvent, preferably 70～80% ethanol aqueous solution therebetween. The weight of the extraction solvent is 1 to 20 times the weight of the medicinal material, preferably 6 to 10 times; the extraction time is 0.5 to 5 hours, preferably 2 hours; the number of extractions is 1 to 5 times, preferably 3 times; the medium used for column chromatography is large Pore adsorption resin, polyamide, reversed-phase silica gel or Sephadex, preferably D101, AB-8, HPD100, HPD300, D1 or D2 type macroporous adsorption resin, the eluting solvent is preferably 30-40% ethanol aqueous solution; step In b), a solvent system composed of fatty ester-fatty alcohol-water or halogenated hydrocarbon-fatty alcohol-water is preferred for high-speed countercurrent chromatography. The fatty ester can be ethyl acetate, butyl acetate, propyl acetate, ethyl formate, Most preferably ethyl acetate, halogenated hydrocarbon can be chloroform, dichloromethane or tetrachloromethane, most preferably chloroform, fatty alcohol can be methanol, ethanol, isopropanol or n-butanol, most preferably methanol, wherein host The rotation speed is 600-1200rpm, preferably 800rpm, the flow rate of the mobile phase pumped into the column is 0.5-5mL/min, preferably 1.5mL/min, the experimental temperature is 10-30°C, preferably 25°C, and the upper phase is fixed phase, the lower phase is the mobile phase; preferably a solvent system composed of fatty alcohol-water-acid or fatty nitrile-water-acid is prepared by high-performance liquid chromatography. The fatty alcohol can be methanol, ethanol, etc., most preferably methanol, fatty nitrile The most preferred is acetonitrile, the acid can be formic acid, acetic acid, phosphate buffer solution, etc., the most preferred is acetic acid, wherein the flow rate is 5-20mL/min, preferably 10mL/min, and the experimental temperature is 10-30°C, preferably 25°C , using gradient elution; due to the instability of caffeoylquinic acid compounds, the separated and collected target components must be recrystallized immediately after vacuum drying, and the recrystallization solvent is an organic solvent or a mixture of water-organic solvents liquid, preferably 80% methanol in water or 80% ethanol in water.
本发明采用高速逆流色谱仪，其根据被分离的物质在两相溶剂系统中的分配系数不同而得到最终分离，这种分离技术由于不使用固体支持介质，避免了因与固体填料发生不可逆吸附而造成的样品损失、失活变性等常规填料易产生的弊端，可使样品得以全部回收，具有分离容量大、性能强、高效、快速的特点。对纯度达不到98％的咖啡酰基奎宁酸类化合物结合高效制备液相色谱进行分离。通过控制实验条件，可以获得高纯度的1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸，5-O-咖啡酰奎宁酸，1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸(纯度大于98％)。本发明方法适合于从各种工艺途径制备的含不同含量的咖啡酰奎宁酸类化合物的粗提物中制备高纯度的1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸，5-O-咖啡酰奎宁酸，1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸单体化合物，适合于各种型号高速逆流色谱仪以及高效制备液相色谱仪制备1-O-咖啡酰奎宁酸，3-O-咖啡酰奎宁酸，4-O-咖啡酰奎宁酸，5-O-咖啡酰奎宁酸，1，3-二-O-咖啡酰奎宁酸，1，5-二-O-咖啡酰奎宁酸，3，5-二-O-咖啡酰奎宁酸，3，4-二-O-咖啡酰奎宁酸和4，5-二-O-咖啡酰奎宁酸单体化合物，能直接使用大量咖啡酰奎宁酸粗提物或其中几种的混合物或其它类似合成物进行进样。The present invention adopts a high-speed countercurrent chromatograph, which obtains the final separation according to the different distribution coefficients of the separated substances in the two-phase solvent system. Since this separation technology does not use a solid support medium, it avoids the occurrence of irreversible adsorption with solid fillers. The disadvantages of conventional fillers such as sample loss and inactivation and denaturation caused by it can make all samples be recovered, and it has the characteristics of large separation capacity, strong performance, high efficiency and fast speed. The caffeoylquinic acid compound whose purity is less than 98% is separated by high performance preparative liquid chromatography. By controlling the experimental conditions, high-purity 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 1,3-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di-O- Caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid (purity greater than 98%). The method of the present invention is suitable for preparing high-purity 1-O-caffeoylquinic acid and 3-O-caffeoylquinic acid from crude extracts containing different contents of caffeoylquinic acid compounds prepared by various processes acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 1,3-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid monomer compounds, suitable for various models Preparation of 1-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid by high-speed countercurrent chromatography and high-efficiency preparative liquid chromatography acid, 1,3-di-O-caffeoylquinic acid, 1,5-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, 3,4-di- O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid monomer compounds can be directly processed using a large amount of caffeoylquinic acid crude extract or a mixture of several of them or other similar compounds Sample.
下面通过实施例及附图进一步说明本发明。必须说明下述实施例是用于说明本发明而不是对本发明的限制。根据本发明的实质对本发明进行简单的改进都属于本发明要求保护的范围。The present invention will be further illustrated below by way of examples and accompanying drawings. It must be stated that the following examples are used to illustrate the present invention and not to limit the present invention. Simple improvements to the present invention according to the essence of the present invention all belong to the protection scope of the present invention.
附图说明Description of drawings
图1为洋蓟粗提物的高效液相色谱图(HPLC)Fig. 1 is the high performance liquid chromatography (HPLC) of artichoke crude extract
图2为本发明的工艺流程图Fig. 2 is a process flow diagram of the present invention
具体实施方式Detailed ways
1.将洋蓟全草于50℃干燥，粉碎。取200g生药，置2000mL圆底烧瓶中，加入75％乙醇1600mL，回流提取3次，每次2h，过滤，合并滤液，减压浓缩至无醇味，回收有机溶剂。将浓缩液进行D101大孔吸附树脂柱层析，先用水洗至无醇味，再依次用20％，40％的乙醇水溶液进行洗脱，收集40％乙醇洗脱液，减压浓缩至无醇味，加入乙醇至含醇量80％，滤去沉淀，取上清液真空干燥得含二咖啡酰奎宁酸类化合物粗品。将粗品进行高速逆流色谱分离纯化，条件为：转速：800rpm；溶剂体系：乙酸乙酯-正丁醇-水(3∶2∶5，v/v)；流速：1.5mL/min；温度：25℃；上相为固定相，下相为流动相；固定相保留率65％。逆流色谱的操作步骤为：将HSCCC溶剂体系按比例配制于分液漏斗中并剧烈振荡，体系分相平衡后分离出上下相，分别超声脱气30min。将固定相泵入高速逆流色谱仪的螺旋管，待螺旋管完全充满后，开启速度控制器，使高速逆流色谱仪按顺时针方向以800rpm旋转，同时以1.5mL/min的流速泵人流动相。当体系达到流体动力学平衡后，将样品由进样阀注入分离管路。柱后流出物以紫外检测器检测。根据色谱图收集色谱峰组分，分离得到九个流份，将各流份合并浓缩，使用分析型高效液相色谱仪进行纯度检测。HPLC分析条件：色谱柱
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